4,4'-[2,2,2-trifluoro-1-(trifluoromethyl)ethylidene]diphenol; bisphenol AF

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 August to 09 November 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Remarks:

Formulation Validation report

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Remarks:

BioAnalytical phase study report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-[2,2,2-trifluoro-1-(trifluoromethyl)ethylidene]diphenol

Cas Number:

1478-61-1

Molecular formula:

C15H10F6O2

IUPAC Name:

4,4'-[2,2,2-trifluoro-1-(trifluoromethyl)ethylidene]diphenol

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at 15-25°C, protected from light
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Formulations were then stirred on a magnetic stirrer to homogenise and aliqoutted.
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: Not stated
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Not stated
- Reactivity of the test material with the incubation material used (e.g. plastic ware): Not stated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final concentration of a dissolved solid, stock liquid or gel: 350, 700, 1400 mg/kg/day

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Hsd:Sprague DawleySD

Details on species / strain selection:

The rat has been selected because there is a large volume of background data in this species. There is also significant further data for this species on the test item being investigated.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo, Blackthorn, UK.
- Sex: DRF phase had 3 males and 3 females per group. Main study phase had 6 males per group
- Age at study initiation: 7-8 weeks
- Weight at study initiation: DRF; 216-235 g (males) or 173-183 g (females) on the first day of dosing. Main study; 220-268 g on the first day of dosing.
- Assigned to test groups randomly: Yes.
- Fasting period before study: No
- Housing: Animals were housed in wire topped, solid bottomed cages, with three animals of the same sex per cage.
- Diet: 5LF2 EU Rodent Diet available ad libitum. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water: Municipal tap water, available ad libitum in water bottles.
- Acclimation period: at least 5 days before commencement of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 25 °C
- Humidity: 40 - 70 %:
- Air changes: Fifteen or more air changes per hour:
- Photoperiod: 12 hours light and 12 hours dark (except during designated procedures)

IN-LIFE DATES: From: 22 Aug 2022 To: 22 Sep 2022

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Arachis Oil
- Justification for choice of solvent/vehicle: Selected as shown to be suitable in previous in vivo studies using the test material and a solubility test
- Concentration of test material in vehicle: 35 - 140 mg/mL (based on dose level of 350 - 1400 mg/kg/day and dose volume of 10 mL/kg).
- Amount of vehicle (if gavage or dermal): Dose Volume of 10 mL/kg.

Details on exposure:

The test material was formulated in Arachis Oil. Test material concentrations were stirred on a magnetic stirrer to homogenise and then aliquoted. All formulations used for animal dosing were protected from light and stored at 15-25°C when not required for dosing. All formulations were used within 2 days of preparation.

Duration of treatment / exposure:

24 hours

Frequency of treatment:

Two administrations, at 0 and 24 hours

Post exposure period:

day 1: Prior to dose, immediate, 1, 2 and 4 hours post dose; day 2: Prior to dose, immediate, 1, 2 and 4 hours post dose*; day 3: Prior to necropsy
(* End of day observations also performed on group 4 animals for welfare reasons.)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle control group (arachis oil)

Doses / concentrations 2

Dose / conc.:

350 mg/kg bw/day

Remarks:

Test. material

Doses / concentrations 3

Dose / conc.:

700 mg/kg bw/day

Remarks:

Test. material

Doses / concentrations 4

Dose / conc.:

1 400 mg/kg bw/day

Remarks:

Test. material

Doses / concentrations 5

Dose / conc.:

20 mg/kg bw/day

Remarks:

Positive control (Cyclophosphamide, CPA)

No. of animals per sex per dose:

DRF phase had 3 males and 3 females per group. Main study phase had 6 animals per group.

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
-Justification for choice of solvent/vehicle: Based on guideline.
Three positive control slides from animals dosed with 20 mg/kg bw cyclophosphamide (prepared under Labcorp study 8487955, Dreher, 2022) were stained and analysed alongside the study slides.

Examinations

Tissues and cell types examined:

Bone Marrow. Initially the relative proportions of polychromatic erythrocytes (PCE), seen as bright orange enucleate cells, and normochromatic erythrocytes (NCE), seen as smaller dark green enucleate cells, were determined until a total of at least 500 cells (PCE plus NCE) had been analysed. Then at least 4000 PCE/animal were examined for the presence of MN.

Details of tissue and slide preparation:

Bone marrow was sampled 48hours after dosing. One femur was removed and cleaned of adherent tissue and the ends removed from the shanks. The bone was flushed with approximately 2 mL of fetal calf serum. The cell suspension was collected and centrifuged at 200 g for 5 min at 15-25°C.

The supernatant was aspirated and discarded. A further 3 mL of foetal bovine serum was added to the tubes followed by gentle resuspension of the cell pellet. The cells were pelleted again and the supernatant aspirated to leave one or two drops and the cell pellet. The pellet was mixed into this small volume of serum in each tube by using a Pasteur pipette, and from each tube one drop of suspension was placed on the end of each of three uniquely labelled slides. A smear was made from the drop by drawing the end of a clean slide along the labelled slide. The slides were immediately stained for 5 minutes in 12.5 µg/mL acridine orange made up in 0.1 M phosphate buffer pH 7.4. Slides were rinsed in phosphate buffer, then dried and stored protected from light at room temperature prior to analysis. Unstained slides were air-dried and initially stored at <-10°C with desiccant.

Evaluation criteria:

A micronucleus test is considered acceptable if it meets the following criteria:
1. Cells must be of normal cell morphology
2. Areas where erythrocytes overlap are ignored
3. A MN must be round or oval in shape
4. A cell containing more than one MN is scored as a single micronucleated cell
5. MN which are refractive, improperly stained or not in the focal plane of the cell are judged to be artefacts and are not scored.

Statistics:

MN PCE in the test article treated groups (Groups 2 to 4) and from the positive control slides were compared against the vehicle control group (Group 1) using ranks of the data via the Wilcoxon Rank Sum Test. The Terpstra-Jonckheere test was conducted to evaluate dose response
A test material is considered positive in the micronucleus test if all of the following criteria are met:
1. A statistically significant increase in the frequency of MN PCE occurred at one or more dose levels
2. The incidence and distribution of MN PCE exceeded the laboratory’s historical vehicle control data
3. A dose-response trend in the proportion of MN PCE was observed.
Positive results in the micronucleus test indicate that a test chemical induces micronuclei, which are the result of chromosomal damage or damage to the mitotic apparatus in the erythroblasts of the test species.

The test article was considered negative in this assay if none of the above criteria were met and bone marrow exposure was confirmed.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF THE DOSE FORMULATION ANALYSIS:

Accuracy:
The concentrations analysed in the dose formulation samples were in agreement with target concentrations (i.e. mean sample concentration results were within or equal to 85%-115%). No test article was detected in the vehicle sample.

RESULTS OF RANGE-FINDING STUDY

- Dose range: 1400 mg/kg bw administered to 3 male and 3 female animals
- Clinical signs of toxicity in test animals: None
- Evidence of cytotoxicity in tissue analysed: N/A
- Rationale for exposure: In a previous single oral dose acute toxicity study the LD50 value of the test item exceeded 2000 mg/kg/day. Clinical signs of toxicity included hunched posture, lethargy, chromodacryorrhoea, rales and piloerection.
- Harvest times: N/A
- High dose with and without activation: N/A
- Other: N/A

RESULTS OF THE BIOANALYSIS PHASE STUDY:

The results of the bioanalysis phase confirm that animals dosed at 350, 700 and 1400 mg/kg/day were systemically exposed to BIS-AF. The highest concentration of test article in blood:water (50:50 v:v) observed in all animals was 1 hour post dose on Day 2 and concentrations generally depleted over time. However, for 2 animals dosed at 350 mg/kg/day (R0107 and R0109) it should be noted that concentrations were lower at the 4 hour post dose time point compared to the 8 hour post dose time point but animal to animal variation can be expected. The accuracy of these results were confirmed during ISR and as such are accepted as valid. There was no test article contamination in the vehicle samples.

RESULTS OF DEFINITIVE STUDY

- Induction of micronuclei (for Micronucleus assay):
No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of test material treated animals compared to the vehicle treated animals.
The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all vehicle control animals was within the historical vehicle control ranges.
Although the MTD was exceeded at 1400 mg/kg (mortality in two animals) the individual micronucleus frequencies of the four remaining animals were comparable with those seen in animals dosed with the vehicle control, such that there was clearly no evidence of micronucleus induction at this dose level.
Cyclophosphamide, the positive control material, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. In addition, the number of micronucleated polychromatic erythrocytes found in the positive control animals was within the historical positive control ranges. Hence, all criteria for an acceptable assay were met.

- Ratio of PCE (for Micronucleus assay):
The animals of the groups, which were treated with test material showed no decrease in the ratio of polychromatic erythrocytes, which indicated a lack of toxic effects of this test material on the erythropoiesis. The animals of the groups treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic erythrocytes, demonstrating toxic effects on erythropoiesis.

- Appropriateness of dose levels and route:
Dose level and route of exposure were selected according to test guideline and guided by a DRF study

Any other information on results incl. tables

Results Summary Table

 

Mean Number of Micronucleated Polychromatic Erythrocytes and Polychromatic Erythrocytes

 

Group	Treatment	Number of Animals	Dose (mg/kg body weight)	Percentage of polychromatic erythrocytes (mean and S.D.)		Percentage of micronucleated polychromatic erythrocytes (mean and S.D.)
	MALES			Mean	SD	Mean	SD
1	Vehicle Control	6	0	43.97	3.56	0.10	0.07
2	Test Material	6	350	45.47	4.49	0.05	0.02
3	Test Material	6	700	48.43	7.78	0.11	0.04
4	Test Material	6	1400	39.25	5.98	0.09	0.07
6	CP	^(1)	20	53.80	3.65	2.84	0.63

Vehicle control = Arachis oil

CP = Cyclophosphamide.

(1)  CPA positive control results were generated using slides from Labcorp study 8487955

(2)  Significantly different from corresponding control group (Wilcoxon Rank Sum test, P>0.05).

Applicant's summary and conclusion

Conclusions:

The test item did not induce micronuclei in the polychromatic erythrocytes of the bone marrow when tested up to 1400 mg/kg/day (a dose deemed to have exceeded the MTD), under the experimental conditions employed.
NB. Awaiting final report pending BioAnalytical phase reporting completion. See attached letter.

Executive summary:

Study Objective 

The objective of this study was to evaluate the potential of the test item to induce micronuclei (MN) in the polychromatic erythrocytes (PCE) of the bone marrow of treated rats.

 

Study Design

The Sprague Dawley rat was the species and strain of choice because it is a readily available rodent which is commonly used for genotoxicity testing, with documented susceptibility to a wide range of toxic materials. Moreover, historical control background data has been generated with this strain.

 

The study procedures described in this report were based on the most recent OECD (29 July 2016) guideline.

 

The test material was a white powder. The test material was prepared in Arachis oil.

 

Based on the results of the dose-range finding study, test concentrations of 1400 mg/kg/day for male animals was selected as maximum dose for the main test due to clinical toxicity signs observed in a previous single oral dose acute toxicity study at concentrations of 2000 mg/kg/day. Based on this information an initial dose of 1400 mg/kg/day was administered in a Range-Finder Experiment. This dose was considered a suitable estimate of the maximum tolerated dose (MTD) Since there were no substantial differences in toxicity between sexes only males were used in the main study.

 

Results of the analyses demonstrated that the achieved concentrations were within 100±15% (with an RSD of ≤10%) of the nominal test article concentrations. The formulations were therefore considered acceptable. No test article was detected in the vehicle sample.

In the main study male animals were dosed two times by oral gavage with vehicle or with 350, 700 and 1400 mg test material per kg body weight at 0 and 24 hours. A positive control group from a separate study was used in this study. In total 4 treatment groups were used, each consisting of 6 animals.

 

In addition, blood for bioanalysis of the test material in plasma was collected from TK animals from all dose groups.

 

In those animals used for bioanalysis (toxicokinetic animals), blood was sampled 0.5, 1, 2 and 4 h after the second dose of either vehicle or the test material. Vehicle dosed animals showed levels below the lower limit of quantification in the plasma. All test material dosed animals showed increased levels of the test material in the plasma, confirming systemic exposure.

 

Approximately 48 hours the animals were sacrificed and bone marrow smears were prepared for micronucleus analysis.

 

Study Results

The results of the bioanalysis phase confirm that animals dosed at 350, 700 and 1400 mg/kg/day were systemically exposed to BIS-AF. 

 

Animals treated with the test material at all doses exhibited group mean %PCE that were similar to the concurrent vehicle control group and which fell within the laboratory’s historical vehicle control data, thus confirming there was no evidence of test article related bone marrow toxicity.

 

Animals treated with the test material at all doses exhibited MN PCE frequencies that were similar to the concurrent vehicle control group and that fell within laboratory's historical vehicle control data. There were no statistically significant increases in micronucleus frequency for any of the groups receiving the test article, compared to the concurrent vehicle control and no evidence of a linear trend.

 

Although the MTD was exceeded at 1400 mg/kg (mortality in two animals) the individual micronucleus frequencies of the four remaining animals were comparable with those seen in animals dosed with the vehicle control, such that there was clearly no evidence of micronucleus induction at this dose level. Given this and the lack of any bone marrow toxicity, together with negative findings at both 350 and 700 mg/kg/day (low and mid dose groups) it is considered to be of sufficient assurance that the micronucleus data were unaffected by toxicity-related artefacts (mortality) and therefore an accurate assessment of micronucleus induction has been achieved from 350 to 1400 mg/kg.

 

 

Conclusions

In conclusion, the test material did not induce micronuclei in the polychromatic erythrocytes of the bone marrow when tested up to 1400 mg/kg/day (a dose deemed to have exceeded the MTD), under the experimental conditions employed.